PROJECT SUMMARY/ABSTRACT In order to survive, animals develop fear responses to dangerous situations. The neural mechanism of learned fear has great survival value for animals, who must predict danger from seemingly neutral contexts. For adaptive fear, the brain discriminates between different contexts and associates only relevant contexts with aversive events. Dysregulation of this process leads to maladaptive overgeneralized fear in PTSD, which affects 7 percent of the U.S. population. A fundamental gap in understanding the mechanism underlying the specificity and persistence of contextual fear memory limits research on developing effective neuromodulatory strategies to prevent long-lasting maladaptive fear in PTSD. The overall objective in this application is to determine the mechanism by which specific contextual fear memory is encoded and consolidated in a network of the hippocampus, neocortex, and amygdala. The rationale for the proposed studies is to fill a critical void in the understanding of fundamental principles of adaptive fear responses to relevant contexts and to provide new insight into developing strategies to attenuate persistent pathological fear in PTSD. The central hypothesis, based on the applicant?s preliminary data, is that (1) the acquisition and consolidation of contextual fear memory require strengthening of engram cell pathways, which connect populations of memory engram cells in a distributed network, and (2) selective weakening of the engram cell pathways prevents conditioned fear responses to a context that predicts danger. This hypothesis will be tested by pursuing three specific aims: (A) Determine how contextual fear memory is encoded in the hippocampal?amygdala circuit and how it is consolidated for permanent storage in prefrontal neocortical circuits (Aims 1 and 2), and (B) Determine input- output connectivity of memory engram cells for contextual fear memory (Aim 3). The first and second aims will investigate synaptic changes in the context-specific hippocampal?amygdala pathway as well as excitatory connections between prefrontal neocortical engram cells during the acquisition and consolidation of contextual fear memory. To accomplish this, the applicant recently developed a novel approach by combining engram cells labeling, optogenetic, and electrophysiological techniques. Under the third aim, engram cells labeling and trans- synaptic tracing will be used to determine how memory engram cells are connected to neurons conveying contextual and aversive signals and neurons generating defensive behavior. The proposed research is innovative because it will use novel combined approaches to efficiently identify the neural correlates of associative memory encoded in functionally heterogeneous neural circuits, which has been unattainable through conventional approaches. The proposed research is significant because it will elucidate how fear memory for a relevant context is encoded and consolidated in a distributed network of memory engram cells and provide insight into developing a novel approach to attenuate chronic maladaptive fear in PTSD without affecting adaptive emotional memories.